Vertebrates are able to produce a vast repertoire of antibody molecules to combat infection. The number of antibody specificities that a human can produce during their lifetime is estimated to be in excess of 109, a number that greatly exceeds the coding capacity of the genome. Instead, the size of the antibody repertoire is the product of gene diversification processes that take place in antibody producing B lymphocytes. The primary B cell repertoire is generated by somatic (V(D)J) recombination in the bone marrow during B cell development. However, this repertoire is neither large enough nor specific enough to provide high affinity antibodies against the range of antigens an animal may encounter. Thus the generation of antibody diversity depends in a major way on secondary diversification processes that occur following V(D)J recombination. Secondary antibody diversification is triggered by deamination of cytidine residues (to yield uracil) within the immunoglobulin locus. This process is catalyzed by the cytidine deaminase AID, which is thought to bind and deaminate ssDNA exposed on the transcribed immunoglobulin gene, generating U:G mismatches that are resolved in a variety of ways to generate point mutations, gene conversion or switch recombination. However, AID-induced uracil lesions can also lead to permanent genomic damage by serving as substrates for chromosome translocations or by mutagenizing non-Ig genes, including oncogenes. Therefore, strict regulation of AID is important for maintaining genomic stability. The long term objective of this proposal is to understand how AID, and by extension antibody diversification, is regulated. This proposal will therefore focus on the following topics: a) the transcriptional regulation of AID (where we propose experiments that will determine the program that leads to induction of AID transcription);b) the regulation of AID at the protein level (where we have used a novel screen to identify the entire set of cellular factors that interact with the deaminase;and also, where we propose detailed studies on one of these cofactors, a protein termed RNF126, which appears to satisfy the requirements of a targeting factor for the deaminase). Somatic hypermutation has been implicated in autoimmune diseases as well as in the generation of B cell lymphomas. Thus the experiments proposed here are important for a better understanding of both autoimmunity and B cell lymphomas. PUBLIC HEALTH RELEVANCE: The experiments proposed here are important for determining how beneficial mutation generates antibody specificities against foreign substances. The mutational process which we propose to study has been implicated in autoimmune diseases as well as in the generation of B cell lymphomas. Understanding the components involved in this process will help us gain better knowledge of the genetic and environmental causes of autoimmunity, as well as the treatment of infectious diseases and tumors.